Chlormadinone acetate (CMA) was administered subcutaneously or orally to the pregnant rats at various gestational stages, and its effects on the course of pregnancy, fetal development and parturition were studied in comparison with those of certain other steroids. The day finding sperm or plug in the vaginal smear was designated as the 1st day of pregnancy. Pregnant rats were isolated from each other, fed with ordinary rat chow, and given tap water ad libitum. The results are summarized as follows :
(1) CMA is as effective as progesterone at a daily dose of 8 mg in maintaining pregnancy of the rat ovariectomized in early pregnancy. Medroxyprogesterone acetate (MAP) is about twice as active as CMA, but norethisterone is inactive in this assay.
(2) Neither progesterone nor CMA inhibits ova-implantation in rats when administered subcutaneously at a daily dose of 1 mg from the first to the sixth day of gestation. MAP produces weak inhibition and norethisterone produces complete inhibition at the same dosage.
(3) Oral administration of CMA produces neither fetal resorption, fetal malformation, fetal masculinization nor feminization in anogenital distance at a daily dose of 8 mg. On the contrary, norethisterone administered subcutaneously at the stage of gestation produces fetal resorption at a daily dose of 2 mg.
(4) When administered subcutaneously at the later stage of gestation, CMA produces feminization of male litters with no masculinization of female litters at a daily dose of 8 mg. MAP induces sexual neutralization, both masculinization and feminization, while norethisterone causes only masculinization of female litters.
(5) Parturition is delayed by the subcutaneous treatments of all progestins from twentieth day of gestation with a daily dose of 1 mg for CMA, 0.4 mg for progesterone and norethisterone, and 0.04 mg for MAP.
Considering the above results, CMA, like progesterone, is characterized as a pregnancy supporting steroid, “gestagen”, whereas norethisterone is considered as a pregnancy blocking steroid, “claudogen”

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For lactation, the adrenal is an indispensable organ, which has been confirmed in hypophysectomized or adrenalectomized animals. However, the problem of concrete relation between these findings in animals and in humans has not been evidenced.
Values of urogenous 17KGS for the first two days, to the complete functions of hypophysis and adrenal in lactating females, were used as control.
On the next day, 20 units ACTH-Z were injected intramusculary twice a day, or 750mg SU-4885 were administered six times every four hours.
Few's (1961) modification method was adapted for the estimation of urogenous 17-ketogenic steroids (11-oxy-17KGS and 11-deoxy-17KGS).
Result as follows ;
(1) Recovery rate of etiocholanolone, mixed group of Comp-s with 17-α-OH-progesterone and 11-β-0H-etiocholanolone, were 93%, 85% and 86% in chromatography, respectively.
(2) Levels of urogenous 17-KGS are correlative with volumes of lactation, strictly speaking, correlative value is not 11-deoxy-17KGS, but 11-Oxy-17KGS.
Volume of milk yield decreased definitely in both cases, 11-oxy-17KGS increased over the normal range abnormally after injection of ACTH-Z or decreased below the basic value after administration of SU-4885. The amount of milk yield increased remarkably in the case which 11-oxy-17KGS increased slightly.
(3) When ACTH-Z was administered on the 6th day after delivery, average amounts of 17KGS reacted with adrenal were 27.4mg, 26.8mg, 9.3mg and 5.6mg on 1st, 2nd, 3rd, and 4th days after administration, respectively. Value of 5.1mg on the 5th day after delivery was used as the control.
Defective females in lactation were classified into higher and lower groups for the reactivity ; the superior females were in middle group.
When doses of 4.5g SU-4885 were administered on the 6th postpartal day, average amounts of 17KGS were 16.2mg and 25.7mg each on that day and the day following administration, and the control was 4.6mg. Both superior and defective groups in lactation reacted normal by administering ACTH-Z and SU-4885.
The relationship between basic excretion levels of 11-oxy-17KGS on the 5th postpartal day and the amount of milk production is correlative : the superior group showed a large amount of 11-oxy-17KGS, and the defective groups showed a lower amount.
(4) Basic excretion level of 17KGS on the 5th postpartal day was lower than that found in pregnancy, and slightly higher than the maximum in non-pregnancy except puerperium.
This shows that the adrenal is hyperfunctional for lactating. Hypophysis and adrenal are stimulated during suckling and this improves the lactation. Disorder of these cycles may cause a reduction in milk production as well as a decrease in basic 17KGS excretion.
(5) Average amounts of 11-deoxy-17KGS and 11-oxy-17KGS excretion when administered ACTH-Z or SU-4885.
Reagent Day after administration 11-Deoxy-17KGS amount percentage mg % 11-Oxy-17KGS amount percentage mg %
ACTH-Z 0 4.46 9.2 33.8 90.8
1 3.7 12.0 26.8 88.0
SU-4885 0 22.6 83.5 3.9 16.5
1 21.04 75.3 6.83 25.7
Control (5 days after delivery) 1.27 17.6 5.93 82.4
(6) There was no relationship between the liver function and the lactation.
The higher ratio of A/G appeared in the defectives. (Amounts of 17KGS, 11-oxy-17KGS and 11-deoxy-17KGS converted into Dehydroisoandrosterone, Tetrahydrocortisone and pregnantriol, respectively.)

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Previous reports provided the confirmatory evidences for the existence of both cellular thyroxine-binding protein (C-T₄BP) and cellular triiodothyronine-binding protein (C-T₃ BP) in rat liver. The present paper describes further investigation concerning C-T₄BP and C-T₃BP by gel filtration on Sephadex G-25 and G-100. The polysaccharide matrix of Sephadex is known to adsorb phenolic compounds such as the thyroid hormones. It would be possible to take advantage of this phenomenon in order to eliminate the thyroid hormones bound nonspecifically to the cellular proteins and, further, to compare the affinity of the hormones for various proteins.
Rat liver soluble proteins containing ¹³¹I-labeled L-thyroxine (T₄-¹³¹I) and L-triiodothyronine (T₃_¹³¹I) were filtered through a Sephadex G-25 column and fractionated with ammonium sulfate. ¹³¹I-labeled hormones bound loosely to liver proteins were eliminated by adsorption to Sephadex gel, and this has clarified the existence of C-T₄BP and C-T₃BP which bound the hormones more tightly. Both the C-T₄BP and the C-T₃BP were found to be precipitated between 0 and 30% saturation of ammonium sulfate.
Rat serum and liver soluble proteins containing T₄-¹³¹I and T₃-¹³¹I were fractionated by gel filtration on Sephadex G-100. Both the C-T₄BP and the C-T₃BP were found to exist in the protein fraction non-diffusible into the gel grains, whereas serum T₄BP emerged in the tailing position of the protein peak corresponding to albumin. These studies indicate the availability for separation of C-T₄BP and C-T₃BP from serum T₄BP and other proteins. This finding also supported the previous suggestion that rat serum T₄BP might not exactly correspond to albumin, and indicated that its molecular size might be a little smaller than that of albumin.
The relative affinities of the thyroid hormones for serum and liver soluble proteins were compared by the use of Sephadex. The affinity of T₃ for serum was much lower than that of T₄whereas the affinity of T₃ for liver soluble proteins was almost equal to that of T₄. This may be due, in part, to the more rapid cellular penetration of T₃ compared to T₄.

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Previous reports described the possibility of the purification of cellular thyroxine-and triiodothyronine binding proteins (C-T₄BP and C-T₃BP) by chromatography on diethylaminoethyl (DEAE) -cellulose, ammonium sulfate precipitation and gel filtration on Sephadex. It is the purpose of the peresent paper to describe the purification by large-scale chromatography on DEAE-cellulose and some properties of C-T₄BP and C-T₃BP from rat liver.
Cellular T₄BP was obtained in a partially purified form by directly chromatographing liver soluble proteins. Cellular T3BP was prepared in a partially purified form by ammonium sulfate precipitation and chromatography on DEAE-cellulose. The positions at which these proteins emerged from the DEAE-cellulose column were in accord with the previous results obtained by small-scale chromatography. Although sedimentation analyses of these preparations showed heterogeneity, the C-T₄BP contained 73% of the protein with a sedimentation coefficient of 5.8S, while the C-T3BP contained 87% of the protein with a sedimentation coefficient of 5.4S. Both the C-T₄BP and the C-T₃BP were found to be non-diffusible into gel grains of Sephadex G-100, and therefore, they were considered to have molecular weights over 100,000.

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Since the action of hormones in the tissue is complicatedly related or combined, the pure action of steroid hormones is rarely investgated in vivo. The purpose of the present examinations was to determine the direct effects of steroid hormones on the multiplication of the following cells :
Fibroblasts from chick embryo hearts, MK cells, PS cells, FL cells, HeLa cells and AH-130 cells. In order to determine accurately the effects, the tissue culture method was employed and a large quantity of the hormones was added in the medium.
1) In 7 days' cultivation, paramethasone showed an inhibitory effect on the multiplication of Fibroblasts from chick embryo hearts proportional to the increase of concentration. Paramethasone in the concentration of 6.6 pg/cc had a 30% inhibitory effect in comparison with the control culture. In the concentration of 6.6 and 33.3μtg/cc, 4-chlorotestosterone acetate gave little effect on the multiplication of Fibroblasts, almost similar to the control culture, but in the concentration of 66.6 μg/cc, it gave slight inhibitory effects.
2) The multiplication of MK cells and PS cells was inhibited by paramethasone in proportion to the increase of concentration : 6.6, 33.3 and 66.6 pg/cc of culture medium, respectively. The inhibitory effects were marked in the case of MK cells.
3) The multiplication of strain FL cells was makedly inhibited by the addition of 66.6, ag/cc paramethasone, and paramethasone in the concentration of 6.6 μg/cc showed 46% inhibitory effect after 7 days' cultivation.
Strain HeLa cells were much more sensitive to paramethasone than strain FL cells, namely, 33.3 μg/cc paramethasone showed 80% inhibitory effect in comparison with the control culture ; 4-chlorotestosterone acetate showed lower inhibitory effect than paramethasone in each concentration : 6.6, 33.3 and 66.6 μg/cc, respectively on the 7th day of cultivation.
4) The inhibitory effects of adrenocorticosteroids on chick embryo pulmonary tissues and FL cells were reversible in a concentration lower than 100 μg per cc.
Paramethasone in the concentration of 66.6 μg/cc had a marked inhibitory effect on the multiplication of FL cells tested, but cell growth was evident after removing paramethasone and thoroughly washing the culture with PBS solution, then adding YLE medium.
5) On examination, the mixture of paramethasone and 4-chlorotestosterone acetate in the concentration of 10μg/cc a slight antagonistic effect was observed. When paramethasone was added alone to the medium, it gave a 50% inhibitory effect on the multiplication of FL cells. In the culture which 10μg/cc paramethasone and 4-chlorotestosterone acetate were added, the inhibitory effect was reduced to 38%.
6) Paramethasone-treated FL cells became round in shape and tended to cohere themselves in sheets in which a great number of giant cells were often seen. These morphologic changes were also seen in high concentration of paramethasone, and FL cells turned into spindle-shaped ones in the concentration of 100 μg/cc.
The nuclei of typical accites hepatoma 130 cells were of various sizes and irregularly shaped. To the contrary, when these cells were treated with paramethasone, they had small and nearly round nuclei in proportion to the increasing concentration, and the cytoplasm contained many vacuoles.
7) In the concentration 50 /2g/cc paramethasone, the mitotic coefficiency decreased to two-thirds of control values in FL cells, five-eighths in MK cells, one-third in AH-130 cells. In 4-chlorotestosterone acetate-treated FL cells, it slightly increased.
8) Prednisolone in the concentration of 10 μg/cc produced a delay and partial inhibition of cytopathic effect when introduced into HeLa cell cultures at the time of infection with HV J. The titer of virus as measured by hemagglutination was dereased slightly in prednisolone-treated groups after 72 hours of incubation.

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Akasu and Tateno (1963) observed in their experiments on rat ascites hepatoma that the lymphnodes, treated with cortisone and eventually atrophied, were seldom attacked with the metastasis of ascites hepatoma, and they assumed the possibility of the arrest of cancer metastasis in man by corticosteroids administration. Previous studies were made to elucidate the mechanism of the above-mentioned observation by means of the determination of the nucleic acid contents of lymphnoid organs.
Under the influence of the implanted tumor (AH 130), untreated rat's lymphnodes swelled in size with the increase of RNA contents and the decline of the ratio of RNA to DNA contents. In treated lymphnodes, however, these changes were not brought about by the implantation of the tumor. Reversely, an increase of DNA contents and a rise of the ratio of RNA to DNA contents were found in treated lymphnodes. This rise of the ratio, induced by cortisone, seemed to be effective for the arrest of the metastasis. The purpose of the present study is to investigate the relation between the nucleic acid contents and the weights of the pelvic lymphnodes excised from cerircal carcinoma patients.
Following lymphadenectomy, the lymphnodes were divided into three groups according to the differences of their weights. Histological examinations were carried out to confirm that these hypertrophied lymphnodes were not yet metastasized from cancer. Throughout this experiment, ribonucleic acids (RNA) were determined by the color reaction of its pentose with orcine and deoxyribonucleic acids (DNA) by the reaction with diphenylamine, after the separation of RNA and DNA by the method based on the Schmidt and Thannhauser procedure.
The results and conclusions of the experiment are summarized as follows :
(1) In comparison with the values of nucleic acid contents in the small pelvic lymphnodes (under 100mg of weights), increase of RNA contents and decline of the ratio of RNA to DNA contents were found unquestionably in the hypertrophied lymphnodes weighing from 100mg to 200mg.
(2) Similar changes have been observed in the experiments of tumor bearing rat lymphnodes as stated above, accordingly the possibility of the same efficacy of corticosteroids administration, discussed in previous paper, may be presumed on the human pelvic lymphnodes.
(3) Clinical application for the inhibition of cancer metastasis after radical treatments may be achieved by proper administration of corticosteroids in man in the future.

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The Metopirone test was carried out on 15 cases of pituitary dwarfism (12 cases idiopathic and 3 cases organic) and 12 cases of non-pituitary dwarfism (5 cases primordial, 4 cases Turner's syndrome and 3 cases with other causes) using the method described in the first part of this study (Folia Endocrinologica Japonica, Vol. 41,683,756, 1965). The following results were obtained. (Fig. 1-5, Table 1-3).
No. of cases examined Normal response Limited pituitary reserve Frank insufficiency
Pituitary (15) (2) (8) (5)
Idiopathic 12 1 8 3
organic 3 1 0 2
Non-pituitary (12) (11) (1) (0)
Primordial 5 5 0 0
Turner's syndrome, 4 4 0 0
Others 1 3 2 1 0
Compared with results obtained by other tests (Table 4a, b), it was concluded that the Metopirone Test was valuable for the early diagnosis of pituitary dwarfism in childhood.

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